Inflammation promotes airway epithelial ATP release via calcium-dependent vesicular pathways.

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.

Mechanosensitive ATP release maintains proper mucus hydration of airways.

The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal autocrine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis.

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia.

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

Computational model for the regulation of extracellular ATP and adenosine in airway epithelia.

Extracellular nucleotides are key components of the signaling network regulating airway clearance. They are released by the epithelium into the airway surface liquid (ASL) to stimulate cilia beating activity, mucus secretion and airway hydration. Understanding the factors affecting their availability for purinoceptor activation is an important step toward the development of new therapies for obstructive lung diseases. This chapter presents a mathematical model developed to gain predictive insights into the regulation of ASL nucleotide concentrations on human airway epithelia. The parameters were estimated from experimental data collected on polarized primary cultures of human nasal and bronchial epithelial cells. This model reproduces major experimental observations: (1) the independence of steady-state nucleotide concentrations on ASL height, (2) the impact of selective ectonucleotidase inhibitors on their steady-state ASL concentrations, (3) the changes in ASL composition caused by mechanical stress mimicking normal breathing, (4) and the differences in steady-state concentrations existing between nasal and bronchial epithelia. In addition, this model launched the study of nucleotide release into uncharted territories, which led to the discovery that airway epithelia release, not only ATP, but also ADP and AMP. This study shows that computational modeling, coupled to experimental validation, provides a powerful approach for the identification of key therapeutic targets for the improvement of airway clearance in obstructive respiratory diseases.

Coupled nucleotide and mucin hypersecretion from goblet-cell metaplastic human airway epithelium.

Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell-associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.

Human alveolar type II cells secrete and absorb liquid in response to local nucleotide signaling.

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na(+) channel-mediated Na(+) absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na(+) channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 µm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

Assessment of extracellular ATP concentrations.

Most cells release ATP to the extracellular milieu. Extracellular ATP plays important signaling roles by activating a score of broadly distributed cell surface purinergic receptors (purinoceptors). Biological responses regulated by purinergic receptors include neurotransmission, smooth muscle relaxation and contraction, epithelial cell ion transport, inflammation, platelet activation, immune responses, cardiac function, endocrine and exocrine secretion, glucose transport, and cell proliferation. ATP concentrations at the cell surface, and consequently the magnitude of purinergic receptor stimulation, reflect a well-controlled balance between rates of ATP release and extracellular metabolism. Given the broad spectrum of responses triggered by extracellular ATP, there is a growing interest in accurately assessing the concentrations of this nucleotide at the cell surface. In this chapter, we discuss the use of the luciferin/luciferase-based reaction to measure extracellular ATP concentrations with high sensitivity. Protocols are adapted to assess ATP levels either in sampled extracellular fluids or in situ at the cell surface. Although our focus is on studies of ATP release from epithelial cells, protocols described here are applicable to practically all cell types.

Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex.

Amongst the many stimuli orienting the growth of plant roots, of critical importance are the touch signals generated as roots explore the mechanically complex soil environment. However, the molecular mechanisms behind these sensory events remain poorly defined. We report an impaired obstacle-avoiding response of roots in Arabidopsis lacking a heterotrimeric G-protein. Obstacle avoidance may utilize a touch-induced release of ATP to the extracellular space. While sequential touch stimulation revealed a strong refractory period for ATP release in response to mechano-stimulation in wild-type plants, the refractory period in mutants was attenuated, resulting in extracellular ATP accumulation. We propose that ATP acts as an extracellular signal released by mechano-stimulation and that the G-protein complex is needed for fine-tuning this response.

17beta-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia.

Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca2+-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl- and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca2+ concentration to stimulate an alternate pathway to Cl- secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate-mediated (UTP-mediated) Cl- secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca2+ influx. This inhibition of Ca2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl- secretion in the airways.

Mathematical model of nucleotide regulation on airway epithelia. Implications for airway homeostasis.

In the airways, adenine nucleotides support a complex signaling network mediating host defenses. Released by the epithelium into the airway surface liquid (ASL) layer, they regulate mucus clearance through P2 (ATP) receptors, and following surface metabolism through P1 (adenosine; Ado) receptors. The complexity of ASL nucleotide regulation provides an ideal subject for biochemical network modeling. A mathematical model was developed to integrate nucleotide release, the ectoenzymes supporting the dephosphorylation of ATP into Ado, Ado deamination into inosine (Ino), and nucleoside uptake. The model also includes ecto-adenylate kinase activity and feed-forward inhibition of Ado production by ATP and ADP. The parameters were optimized by fitting the model to experimental data for the steady-state and transient concentration profiles generated by adding ATP to polarized primary cultures of human bronchial epithelial (HBE) cells. The model captures major aspects of ATP and Ado regulation, including their >4-fold increase in concentration induced by mechanical stress mimicking normal breathing. The model also confirmed the independence of steady-state nucleotide concentrations on the ASL volume, an important regulator of airway clearance. An interactive approach between simulations and assays revealed that feed-forward inhibition is mediated by selective inhibition of ecto-5'-nucleotidase. Importantly, the model identifies ecto-adenylate kinase as a key regulator of ASL ATP and proposes novel strategies for the treatment of airway diseases characterized by impaired nucleotide-mediated clearance. These new insights into the biochemical processes supporting ASL nucleotide regulation illustrate the potential of this mathematical model for fundamental and clinical research.

Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells.

The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca(2+)-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca(2+)-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca(2+)-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y(2) receptor-stimulated increase of cytosolic Ca(2+) concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL.

Physiological regulation of ATP release at the apical surface of human airway epithelia.

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells.

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b-) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (-/-)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (-/-) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (-/-) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (-/-) and WT mice after luminal hypotonic challenge, but VDAC-1 (-/-) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (-/-) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

Nucleotide release provides a mechanism for airway surface liquid homeostasis.

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.